连翘酯苷对传染性支气管炎病毒感染鸡toll受体7信号通路调控

Infected by infectious bronchitis virus(IBV), chicken recognize and initialize immunity and inflammation by toll like receptor 7 sianaling pathway. Forsythiaside is effective for inhibition of infectious bronchitis virus, but the detailed pharmacological mechanism is still unknown. Does forsythiaside control IBV by toll like receptor 7 signaling pathway? What is the target protein? Using cell culture in vitro, RNAi of toll like receptor 7 and MyD88, adding cross talk inhibtor(RIP1), special inhibitor, agonist and so on,the effect on toll like receptor 7, nuclear factor κB, interferon regulation factor 7 signaling pathway, and production of cell immunity-proinflammatory(Interleukins, Interferon, Tumor necrosis factors) factors induced by forsythiaside would be confirmed,and its target protein receptor will be validated. These effects on virus replication will also be determined. At last, the pharmacological mechanism will be validated by challenge special pathagen free chicken with infectious brochitis virus. the morphology and pathology, cell ultra microstructure, inflamation, immunity, virus capacity, and the special antibody titer in target tissues,blood and immune organs will be compared. In all, the toll like receptor 7 signaling pathway for forsythiaside will be comprehensively studied through three profiles of drug, virus and cell- organism interaction, and two sides of correct and reverse.

鸡体toll受体7识别鸡传染性支气管炎病毒感染，并介导免疫和炎性反应相关信号通路。连翘酯苷是否通过调控toll样受体7信号通路防治鸡传支病？其作用的靶蛋白是什么？本课题拟利用体外细胞培养，对toll受体7信号通路采用RNA干扰、交汇信号通路抑制剂、独特性抑制剂、受体激动剂等处理，探究连翘酯苷对鸡传支病毒感染细胞的toll样受体7、干扰素调节因子7、干扰素调节因子3、核因子KB(NF-kB)信号通路作用，对细胞免疫炎性因子（白介素、干扰素、肿瘤坏死因子）产生，及上述调控作用对鸡传支病毒复制的影响。最后，利用SPF鸡感染传染性支气管炎病毒模型，研究连翘酯苷对病毒靶组织和免疫器官组织病理、超微结构、免疫和炎性反应、病毒载量、特异性抗体产生等作用。从连翘酯苷-体外细胞和动物机体-传支病毒多层次，正反两方面研究连翘酯苷防治鸡传支作用toll样受体信号通路作用机制

研究连翘酯苷A对SPF雏鸡感染IBV的药效。检测肺、支气管、脾、肾等组织病理变化和TLR7信号通路相关（TLR7，NFkB，TRAF6，MyD88， IIRF3, MDA5, LGP2, 和NLRC5）细胞因子表达，炎性细胞因子(IFN-α, TNF-α, IL-1β）的表达和IBV病毒载量，ELISA方法检测血清中TNF-α，IL-1β，IBV抗体含量和血生化指标，表明连翘酯苷调控IBV感染激活IRF7/NF-KB信号通路，诱生IFN，TNF-α，IL-1β和抗体水平。减轻IBV感染造成的组织损伤，缩短病程。. 建立IBV感染鸡传代细胞系DF-1、HD11细胞感染模型，荧光定量PCR和Western-blot检测细胞内IBV N基因及抗病毒基因IRF7、IFN-α、IFNβ、Mx、PKR、OAS mRNA，以及NF-κB及其下游相关的基因的表达，已感细胞进行连翘酯苷A药物治疗处理，连翘酯苷A显著提高细胞内受体（MDA5，LGP２，NLRC5）和抗病毒基因（IRF７和NF－κB1的表达在感染后显著升高，IFN－α，IFN－β）ｍRNA表达。. 连翘酯苷A通过激活IRF7/NF-KB发挥抗IBV作用。分别构建pEGFPNF-κB1，pEGFP-N3-IRF7过表达质粒并转染细胞，采用IRF7 siRNA和NF-κB抑制剂PDTC，研究IRF7和NF-κB在抗IBV中的作用。1）荧光定量 PCR检测细胞内IBV N基因和抗病毒相关基因IRF7、IFN-α、IFN-β、Mx、PKR、OAS mRNA水平，结果IRF7 siRNA 抑制IFN信号通路，IBV复制增加，连翘酯苷A缓解IRF7 siRNA的作用；pEGFP-N3-IRF7表达质粒组IRF7表达增加，激活IFN信号通路，抑制IBV复制；加入连翘酯苷A协同pEGFP-N3-IRF7的作用。表明连翘酯苷可能是通过激活IRF7进而启动IFN信号通路发挥抗IBV作用的。2）PDTC抑制NF-κB表达，IL-1β、TNF-α、IL-8表达明显降低，细胞内IBV载量升高；同时，连翘酯苷A逆转NF-KB抑制剂作用。pEGFPNF-κB1组IL-1β、TNF-α、IL-8表达明显升高，细胞内IBV载量下降；同时，连翘酯苷A协同NF-KB发挥抗病毒作用。